The present invention relates to neuron-restrictive silencer factor proteins, nucleic acids, and antibodies thereto.
The molecular basis of neuronal determination and differentiation in vertebrates is not well understood. It other lineages, systematic promoter analysis of cell-type specific genes has led to the identification of genetically essential transcriptional regulators of lineage determination or differentiation L. M. Corcoran, et al., Genes and Development 7, 570-582 (1993); S. Li, et al., Nature (London) 347, 528-533 (1990); L. Pevny, et al., Nature 349, 257-260 (1991). To apply this approach to the development of neurons, the transcriptional regulation of a neuron-specific gene, SCG10, has been previously examined (D. J. Anderson, R. Axel, Cell 42, 649-662 (1985). SCG10 is a 22 Kd, membrane-associated phosphoprotein that accumulates in growth cones and is transiently expressed by all developing neurons (R. Stein, N. Mori, K. Matthews, L.-C. Lo, D. J. Anderson, Neuron 1, 463-476 (1988); U. K. Shubart, M. D. Banerjce, J. Eng. DNA 8, 389-398 (1989)). Upstream regulatory sequences controlling SCG10 transcription have been analyzed using promoter fusion constructs, both in transient cell transfection assays and in transgenic mice (N. Mori, R. Stein, O. Sigmund, D. J. Anderson, Neuron 4, 583-594 (1990); C. W. Wuenschell, N. Mori, D. J. Anderson, Neuron 4, 595-602 (1990)). These studies revealed that the 5xe2x80x2flanking region can be functionally separated into two regulatory domains: a promoter-proximal region that is active in many cell lines and tissues, and a distal region that selectively represses this transcription in non-neuronal cells. Deletion of the distal region relieves the repression of SCG10 transgenes in non-neuronal tissues, such as liver, in transgenic mice (C. W. Wuenschell, N. Mori, D. J. Anderson, Neuron 4, 595-602 (1990); D. J. Vandenbergh, C. W. Wuenschell, N. Mori, D. J. Anderson, Neuron 3, 507-518 (1989)). Furthermore, in transient cell transfection assays this distal region could repress transcription from a heterologous promoter in an orientation- and distance-independent manner (N. Mori, R. Stein, O. Sigmund, D. J. Anderson, Neuron 4, 583-594 (1990)), satisfying the criteria for a silencer: a sequence analogous to an enhancer but with an opposite effect on transcription (A. H. Brand, L. Breeden, J. Abraham, R. Sternglanz, K. Nasmyth, Cell 41, 41-48 (1985)). The finding that neuron-specific gene expression is controlled primarily by selective silencing stands in contrast to most cell type-specific genes studied previously, in which specificity is achieved by lineage-specific enhancer factors (T. Maniatis, S. Goodbourn, J. A. Fischer, Science 236, 1237-1245 (1987); P. Mitchell, R. Tjian, Science 245, 371-378 (1989); P. F. Johnson, S. L. McKnight, Annu. Rev. Biochem. 58, 799-839 (1989); X. He, M. G. Rosenfeld, Neuron 7, 183-196 (1991)).
A detailed analysis of the SCG10 silencer region identified a ca. 24 bp element necessary and sufficient for silencing (N. Mori, S. Schoenherr, D. J. Vandenbergh, D. J Anderson, Neuron 9, 1-10 (1992)). Interestingly, similar sequence elements were identified in two other neuron-specific genes: the rat type II sodium (NaII) channel and the human synapsin 1 genes (N. Mori, S. Schoenherr, D. J. Vandenbergh, D. J Anderson, Neuron 9, 1-10 (1992); R. A. Maue, S. D. Knaner, R. H. Goodman, G. Mandel, Neuron 4, 223-231 (1990); S. D. Kraner, J. A. Chong, H. J. Tsay, G. Mandel, Neuron 9, 37-44 (1992); L. Li, T. Suzuki, N. Mori, P. Greengard, Proceedings of the National Academy of Science (USA) 90, 1460-1464 (1993)). These sequence elements were shown to possess silencing activity in transfection assays as well, and has been named the neuron-restrictive silencer element (NRSE) (N. Mori, S. Schoenherr, D. J. Vandenbergh, D. J Anderson, Neuron 9, 1-10 (1992)); in the context of the Nail channel gene, it has also been called repressor element 1 (RE1) (S. D. Kraner, J. A. Chong, H. J. Tsay, G. Mandel, Neuron 9, 37-44 (1992)).
Using electrophoretic mobility shift assays, the NRSEs in the SCG10, NaII channel and synapsin I genes were all shown to form complexes with a protein(s) present in non-neuronal cell extracts, but absent in neuronal cell extracts (Mori et al., supra), Kraner et al., supra, Li et al., supra). This protein was termed the neuron-restrictive silencer factor (NRSF). Both the SCG10 and the NaII channel NRSEs competed with similar efficacy for NRSF, suggesting that this protein could bind both NRSEs (Mori et al., supra). Moreover, mutations in the NRSE that abolished NRSF binding in vitro eliminated the silencing activity of the NRSE in transient transfection assays. These data implicated NRSF in the lineage-specific repression of at least two neuron-specific genes.
The present invention provides recombinant NRSF proteins, and isolated or recombinant nucleic acids which encode the NRSF proteins. Also provided are expression vectors which comprise nucleic acid encoding an NRSF protein operably linked to transcriptional and translational regulatory nucleic acid, and host cells which contain the expression vectors.
An additional aspect of the present invention provides methods for producing NRFS proteins which comprise culturing a host cell transformed with an expression vector and causing expression of the nucleic acid encoding the NRSF protein to produce a recombinant NRSF protein.
An additional aspect provides antibodies to the NRSF proteins of the present invention.